Plant seed oils are major sources of essential polyunsaturated fatty acids for human diets and renewable feedstocks for chemical industries. The enzymes of the fatty acid synthase complex in the plastids of developing seeds are responsible for the biosynthesis of fatty acids that are channeled into the cytosolic acyl-CoA pool to sustain triacylglycerol accumulation. Triacylglycerol (TAG) biosynthesis is located in the endoplasmic reticulum with glycerol-3-phosphate and fatty acyl-CoAs as the primary substrates. There are three acyltransferases involved in the plant storage lipid bioassembly, namely the glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), the lyso-phosphatidic acid acyltransferase (LPAT, EC 2.3.1.51) and the diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). These three acyltransferases catalyze the stepwise acylation of the glycerol backbone with the final step being the acylation of sn-1, 2-diacylglycerol (DAG) by DGAT into the formation of TAGs, a biochemical process generally known as the Kennedy pathway (Stymne and Stobart, 1987).
Among the three ER-based fatty acyl-CoA acyltransferases, only LPAT gene(s) have been cloned from plants (Knutzon et al., 1995, Lassner et al., 1995). Like several other enzymes involved in storage lipid biosynthesis, acyltransferases are intrinsic ER proteins and are extremely difficult to purify. The research on plant DGAT has been largely limited to studies of activity profiles by using the particulate fractions generated by differential centrifugation of seed or microspore-derived embryo homogenates (Weselake et al., 1993). Although partial purification of DGAT from cotyledons of germinating soybean seeds was reported (Kwanyuan and Wilson, 1988), detailed molecular characterization of this enzyme is lacking.
Reference is made to R61u012 Database Entry Ac005917; Accession number AC005917; 4 Nov. 1998; LIN X. ET AL.: “Arabidopsis thaliana chromosome II section 113 of 255 of the complete sequence”. This relates to a nucleotide sequence deposit first submitted to the NCBI GenBank on Nov. 3, 1998 without any identification of putative coding sequences contained therein. There was no reference in the deposited materials to a “diacylglycerol O-acyltransferase gene”.
Reference is also made to VESNA KATAVIC ET AL.: “Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity”; PLANT PHYSIOLOGY, vol. 108, 1995, pp. 399–409. This reference discloses an Arabidopsis mutant designated AS11 that has reduced diacylglycerol acyltransferase activity. The reference does not disclose any DNA sequences whatsoever and teaches only that alterations in DGAT activity may lead to changes in fatty acid content.
Accordingly, while the Kennedy pathway is known and shows the steps in the biosynthesis of TAGs in plants, there has not been any identification and use of a genetic element that can be used reliably in plants to modify TAG synthesis and composition in a way that may be exploited commercially.